As one of the top public health concerns of the 21st century, obesity remains only partly understood from a mechanistic basis. Another health concern of equal importance is disease cachexia, which is characterized by a significant decrease in appetite leading to muscle atrophy in a person who is not actively pursuing weight loss. The central melanocortin system is known to play a regulatory role in energy metabolism, on both obesity and disease cachexia. The melanocortin-4-receptor (MC4R), a component of this system, is a seven- transmembrane G-protein coupled receptor that upon activation is known to cause a decrease in feeding, while antagonizing the receptor causes ravenous feeding without behavioral training. Because of this, MC4R is a target for drug development in the treatment of obesity and cachexia. Our lab recently discovered a novel signaling pathway in which MC4R couples to an inwardly rectifying potassium channel, Kir7.1, upon activation, leading to a decrease in feeding in vivo. Multiple obesity-associated mutations in the MC4R gene as well as the use of various MC4R-specific pharmacological compounds have suggested that there may be alternative pathways by which MC4R signaling occurs. Thus, we hypothesize that MC4R exhibits biased signaling in the regulation of feeding and body weight, and testing this hypothesis is the focus of this application. Utilizing the innovative clustered, regularly interspaced, short palindromic repeats (CRISPR) technology, we will introduce precise modifications to the genome of experimental animals that will allow us to identify the site(s) and mode(s) of MC4R signaling in vivo. As such, we have proposed two aims to address our research question: (1) characterization of the biased signaling of novel obesity-associated mutations of MC4R and (2) identification of the neuroanatomical distribution of MC4R and Kir7.1 signaling in vivo. Through this training plan I will gain expertise in cutting-edge genetic techniques, GPCR and channel pharmacology, and small animal laboratory practices. Training in these studies will be paired with educational work in broader aspects of obesity treatment and prevention to formulate a holistic approach to treating and preventing obesity.